Measuring cAMP and Ca2+ in Opsin-HEK Cells

The intracellular cAMP and Ca²⁺ levels in opsin-expressing HEK293S cells were measured using the GloSensor cAMP assay and the aequorin assay, respectively, as described previously (22 (link), 26 (link), 69 (link), 70 (link)). The pGloSensor-22F cAMP plasmid (Promega) was used for the GloSensor cAMP assay. The wild-type aequorin obtained by introducing two reverse mutations into the plasmid [pcDNA3.1+/mit-2mutAEQ] (Addgene #45539) (71 (link)) was used for the aequorin assay. A broadband green LED light and narrow-band violet (410 nm) and blue (430 nm) LED lights were applied for 5 s in the GloSensor cAMP assay and for 1 s in the aequorin assay as light stimuli.

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Koyanagi M., Shen B., Nagata T., Sun L., Wada S., Kamimura S., Kage-Nakadai E, & Terakita A. (2022). High-performance optical control of GPCR signaling by bistable animal opsins MosOpn3 and LamPP in a molecular property–dependent manner. Proceedings of the National Academy of Sciences of the United States of America, 119(48), e2204341119.

Publication 2022

pGloSensor-22F cAMP plasmid pcDNA3.1+/mit-2mutAEQ

Aequorin Assay Cells Intracellular Light Mutations Opsin Plasmid Promega Violet

Corresponding Organization :

Other organizations : Osaka City University

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Variable analysis

independent variables

Light stimuli: Broadband green LED light, narrow-band violet (410 nm) and blue (430 nm) LED lights

dependent variables

Intracellular cAMP levels in opsin-expressing HEK293S cells (measured using the GloSensor cAMP assay)
Intracellular Ca2+ levels in opsin-expressing HEK293S cells (measured using the aequorin assay)

control variables

Opsin-expressing HEK293S cells (used as the experimental system)
GloSensor cAMP assay (used to measure intracellular cAMP levels)
Aequorin assay (used to measure intracellular Ca2+ levels)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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